Why has our culture medium suddenly become toxic?!?

This week we're wrestling with a practical mystery - for unknown reasons the 'brain heart infusion' culture medium we usually use for H. influenzae no longer supports growth.

The problem first surfaced on Monday, when one of the post-docs found that cultures she had inoculated the night before had no growth. Suspecting that she'd made a mistake, she carefully reinoculated them, only to find no growth again on Tuesday morning. On Wednesday another post-doc found that her cultures hadn't grown either.

The medium had been prepared by our new lab assistant, so naturally we wondered if she'd made a mistake. This seemed unlikely because (1) she's very careful, and (2) making it is very simple (just dissolve 37 g of BHI powder in 1 liter of water, pour it into bottles and autoclave, and (3) the medium looked pretty normal (clear, golden brown) and we had a hard time imagining what could entirely prevent bacteria from growing in it. When she came in she confirmed that the medium had been made from the nearly-empty container of Difco BHI powder we've been using for months now, not the other-brand bottle we were going to try out someday soon.

A measuring error might have given less growth, but not none. Making the medium up with the 'wrong' water shouldn't have mattered - I'm pretty sure cells would grow fine even if the medium was made with plain tap water instead of the distilled water we meticulously use. The usual supplements of hemin and NAD were from stock tubes that had worked fine in previous cultures.

Simple tests didn't find a problem - the BHI smelled normal, and had a reasonable pH of about 8. The post-docs tried inoculating E. coli into it (less fussy than H. influenzae), but these cells didn't grow either. As well as inoculating the E. coli into 5ml of BHI in our usual culture tubes they also cleverly tried inoculating it directly into the stock bottle, and surprisingly these cells grew fine. So they suspected that maybe the problem was with the culture tubes rather than the BHI. In a parallel test they had made a fresh batch of BHI, and tested this in culture tubes with H. influenzae and E. coli. This time the E. coli again didn't grow, but the H. influenzae did, again directing suspicion to the culture tubes.

They did some more tests yesterday, so maybe today we'll figure out what the problem is...